The time-zero HSQC method improves the linear free energy relationship of a polypeptide chain through the accurate measurement of residue-specific equilibrium constants.

EXSY (exchange spectroscopy) NMR provides the residue-specific equilibrium constants, K, and residue-specific kinetic rate constants, k, of a polypeptide chain in a two-state exchange in the slow exchange regime. A linear free energy relationship (LFER) discovered in a log k versus log K plot is considered to be a physicochemical basis for smooth folding and conformational changes of protein molecules. For accurate determination of the thermodynamic and kinetic parameters, the measurement bias arising from state-specific differences in the R1 and R2 relaxation rates of 1H and other nuclei in HSQC and EXSY experiments must be minimized. Here, we showed that the time-zero HSQC acquisition scheme (HSQC0) is effective for this purpose, in combination with a special analytical method (Π analysis) for EXSY. As an example, we applied the HSQC0 + Π method to the two-state exchange of nukacin ISK-1 in an aqueous solution. Nukacin ISK-1 is a 27-residue lantibiotic peptide containing three mono-sulfide linkages. The resultant bias-free residue-based LFER provided valuable insights into the transition state of the topological interconversion of nukacin ISK-1. We found that two amino acid residues were exceptions in the residue-based LFER relationship. We inferred that the two residues could adopt special conformations in the transition state, to allow the threading of some side chains through a ring structure formed by one of the mono-sulfide linkages. In this context, the two residues are a useful target for the manipulation of the physicochemical properties and biological activities of nukacin ISK-1.

Residue-based correlation between equilibrium and rate constants is an experimental formulation of the consistency principle for smooth structural changes of proteins.

The consistency principle represents a physicochemical condition requisite for ideal protein folding. It assumes that any pair of amino acid residues in partially folded structures has an attractive short-range interaction only if the two residues are in contact within the native structure. The residue-specific equilibrium constant, K, and the residue-specific rate constant, k (forward and backward), can be determined by NMR and hydrogen-deuterium exchange studies. Linear free energy relationships (LFER) in the rate-equilibrium free energy relationship (REFER) plots (i.e., log k vs. log K) are widely seen in protein-related phenomena, but our REFER plot differs from them in that the data points are derived from one polypeptide chain under a single condition. Here, we examined the theoretical basis of the residue-based LFER. First, we derived a basic equation, ρij=½(φi+φj), from the consistency principle, where ρij is the slope of the line segment that connects residues i and j in the REFER plot, and φi and φj are the local fractions of the native state in the transient state ensemble (TSE). Next, we showed that the general solution is the alignment of the (log K, log k) data points on a parabolic curve in the REFER plot. Importantly, unlike LFER, the quadratic free energy relationship (QFER) is compatible with the heterogeneous formation of local structures in the TSE. Residue-based LFER/QFER provides a unique insight into the TSE: A foldable polypeptide chain consists of several folding units, which are consistently coupled to undergo smooth structural changes.

Structural and functional analysis of hyper-thermostable ancestral L-amino acid oxidase that can convert Trp derivatives to D-forms by chemoenzymatic reaction.

Production of D-amino acids (D-AAs) on a large-scale enables to provide precursors of peptide therapeutics. In this study, we designed a novel L-amino acid oxidase, HTAncLAAO2, by ancestral sequence reconstruction, exhibiting high thermostability and long-term stability. The crystal structure of HTAncLAAO2 was determined at 2.2 Å by X-ray crystallography, revealing that the enzyme has an octameric form like a "ninja-star" feature. Enzymatic property analysis demonstrated that HTAncLAAO2 exhibits three-order larger kcat/Km values towards four L-AAs (L-Phe, L-Leu, L-Met, and L-Ile) than that of L-Trp. Through screening the variants, we obtained the HTAncLAAO2(W220A) variant, which shows a > 6-fold increase in kcat value toward L-Trp compared to the original enzyme. This variant applies to synthesizing enantio-pure D-Trp derivatives from L- or rac-forms at a preparative scale. Given its excellent properties, HTAncLAAO2 would be a starting point for designing novel oxidases with high activity toward various amines and AAs.

Retrospective study for the universal applicability of the residue-based linear free energy relationship in the two-state exchange of protein molecules.

Multiprobe measurements, such as NMR and hydrogen exchange studies, can provide the equilibrium constant, K, and rate constants for forward and backward processes, k and k', of the two-state structural changes of a polypeptide on a per-residue basis. We previously found a linear relationship between log K and log k and between log K and log k' for the topological exchange of a 27-residue bioactive peptide. To test the general applicability of the residue-based linear free energy relationship (rbLEFR), we performed a literature search to collect residue-specific K, k, and k' values in various exchange processes, including folding-unfolding equilibrium, coupled folding and binding of intrinsically disordered peptides, and structural fluctuations of folded proteins. The good linearity in a substantial number of the log-log plots proved that the rbLFER holds for the structural changes in a wide variety of protein-related phenomena. Among the successful cases, the hydrogen exchange study of apomyoglobin folding intermediates is particularly interesting. We found that the residues that deviated from the linear relationship corresponded to the α-helix, for which transient translocation had been identified by other experiments. Thus, the rbLFER is useful for studying the structures and energetics of the dynamic states of protein molecules.

Residue-Specific Kinetic Insights into the Transition State in Slow Polypeptide Topological Isomerization by NMR Exchange Spectroscopy.

The characterization of the transition state is a central issue in biophysical studies of protein folding. NMR is a multiprobe measurement technique that provides residue-specific information. Here, we used exchange spectroscopy to characterize the transition state of the two-state slow topological isomerization of a 27-residue lantibiotic peptide. The exchange kinetic rates varied on a per-residue basis, indicating the reduced kinetic cooperativity of the two-state exchange, as well as the previously observed reduced thermodynamic cooperativity. Furthermore, temperature-dependent measurements revealed large variations in the activation enthalpy and entropy terms among residues. Interestingly, we found a linear relationship between the logarithm of the equilibrium constants and that of the exchange rates. Because the data points are derived from amino acid residues in one polypeptide chain, we refer to the linear relationship as the residue-based linear free energy relationship (rbLFER). The rbLFER offers information about the transition state of the two-state exchange.

Crystal structures of the archaeal RNase P protein Rpp38 in complex with RNA fragments containing a K-turn motif.

A characteristic feature of archaeal ribonuclease P (RNase P) RNAs is that they have extended helices P12.1 and P12.2 containing kink-turn (K-turn) motifs to which the archaeal RNase P protein Rpp38, a homologue of the human RNase P protein Rpp38, specifically binds. PhoRpp38 from the hyperthermophilic archaeon Pyrococcus horikoshii is involved in the elevation of the optimum temperature of the reconstituted RNase P by binding the K-turns in P12.1 and P12.2. Previously, the crystal structure of PhoRpp38 in complex with the K-turn in P12.2 was determined at 3.4 Šresolution. In this study, the crystal structure of PhoRpp38 in complex with the K-turn in P12.2 was improved to 2.1 Šresolution and the structure of PhoRpp38 in complex with the K-turn in P12.1 was also determined at a resolution of 3.1 Å. Both structures revealed that Lys35, Asn38 and Glu39 in PhoRpp38 interact with characteristic G·A and A·G pairs in the K-turn, while Thr37, Asp59, Lys84, Glu94, Ala96 and Ala98 in PhoRpp38 interact with the three-nucleotide bulge in the K-turn. Moreover, an extended stem-loop containing P10-P12.2 in complex with PhoRpp38, as well as PhoRpp21 and PhoRpp29, which are the archaeal homologues of the human proteins Rpp21 and Rpp29, respectively, was affinity-purified and crystallized. The crystals thus grown diffracted to a resolution of 6.35 Å. Structure determination of the crystals will demonstrate the previously proposed secondary structure of stem-loops including helices P12.1 and P12.2 and will also provide insight into the structural organization of the specificity domain in P. horikoshii RNase P RNA.

Central retinal vein occlusion caused by human herpesvirus 6.

We describe a 1-year-old girl with central retinal vein occlusion caused by human herpesvirus 6. She received systemic corticosteroid therapy. Although there was retinal recovery after the therapy, the visual acuity in her left eye still could not be measured.

[Comparison of viewpoints and awareness between patients with and without a "family pharmacy"].

To evaluate differences in viewpoints and awareness between patients with and without a "family pharmacy", we performed a questionnaire survey. The questionnaire consisted of 21 questions including "I often read books and journals about health." and "Patients have the right of being informed of the medical treatment they receive". The degree of applicability was selected among 6 grades ("definitely inapplicable"--"definitely applicable"). The subjects were classified into 3 groups "without a family pharmacy", "with a regular pharmacy for each hospital/clinic" and "with a family pharmacy", and factor analysis was performed. As a result, 5 factors were extracted in each group. However, the order of the 2nd-4th factors differed among the 3 groups. Paying attention to these differences, we found that patients "with a family pharmacy" have resolved doubts about or dissatisfaction with medical care to some extent.

Collection of medical drug information in pharmacies: Drug Event Monitoring (DEM) in Japan.

To establish a system for collecting and reporting information from community pharmacists such as that on adverse effects, the Japan Pharmaceutical Association (JPA) conducts Drug Event Monitoring (DEM). In the fiscal year 2002, a survey was carried out to clarify the incidence of sleepiness due to antiallergic drugs. The investigated active ingredients were ebastine, fexofenadine hydrochloride, cetirizine hydrochloride, and loratadine. Community pharmacists asked the following question to patients who visited their pharmacies: "Have you ever become sleepy after taking this drug?" During a 4-week survey period, reports of 94256 cases were collected. To evaluate the incidence of sleepiness, we analyzed cases in which reports showed alleged absence of concomitant oral drugs, and drug use in conformity with the dose and method described in package inserts. The incidence of sleepiness was significantly different among the drugs (chi(2)-test, p<0.001). The observed incidences of sleepiness due to the drugs (8.8-20.5%) were higher than those described in each package insert (1.8-6.35%). This may be because an active question was used ("Have you ever become sleepy after taking this drug?"). Active intervention by pharmacists may be useful for collecting more information on improvement in the QOL of patients and safety. In addition, the pharmacists were asked to report events other than "sleepiness" in the free description column of the report. Some symptoms not described in the package inserts were reported, suggesting that DEM may lead to the discovery of new adverse effects. These results suggest that community pharmacists have a good opportunity to collect information in DEM, and safety information such as that on adverse effects can be obtained from pharmacies.

[Classification of pharmaceutical services from the viewpoint of patient satisfaction/dissatisfaction].

A survey was conducted to examine what the public expects of pharmacists. We created 26 questions based on 26 different situations that patients encounter at pharmacies in order to assess patient satisfaction/dissatisfaction. Some questions were designed to compare pairs of symmetrical situations. The survey was carried out in February and March 2001. The surveys were conducted with patients and/or patients' relatives who brought their prescriptions to pharmacies and with employees of pharmaceutical companies and their family members. A factor analysis extracted two factors among the 26 variables, the first being patient dissatisfaction and the second being patient satisfaction. However, factor loadings for some paired situations were not necessarily symmetrical and thus the absolute values of the scores were not equal. The results suggest that satisfaction on one side does not entail dissatisfaction on the other side and thus satisfaction and dissatisfaction can be examined as separate and distinct entities. Based on these results, we classified pharmaceutical services into two types. The first type is referred to as instrumental service. In this situation patients do not feel great satisfaction even if such service is provided. However, they feel great dissatisfaction if the service is not provided. The second type is referred to as expressive service. These patients feel some satisfaction if such service is provided, however, they do not feel a fatal dissatisfaction if the service is not provided. Our research documents that when examining data based on the assumption that there is a different dimensionality for patient satisfaction and dissatisfaction, it is possible to gain insights into new aspects of pharmaceutical services that are otherwise impossible to assess.

[The expected role of community pharmacist in society].

With recent advances in the separation of dispensing and prescribing drugs (Bungyo), the social role of community pharmacists has expanded. Therefore we carried out a questionnaire survey on the "image of community pharmacists" among patients/the public, physicians, and community pharmacists. The understanding of their role by community pharmacists differed slightly from that by patients/the public or physicians. Better mutual understanding among the three parties is necessary for smooth communication. Dispensing and handing over drugs to patients are not the only tasks required of community pharmacists. Evaluation of records of telephone drug counseling by the Japan Pharmaceutical Association revealed that many callers desired not only drug information but also the opinions and judgments of pharmacists. Providing drug information is an important task for community pharmacists. In addition, counseling on anxiety and worries about health may be necessary. Bungyo has also affected drug distribution. In this field, the role of community pharmacists in providing drug information may markedly change in the future.

Patient perspectives on provision of drug information services in Japan.

Today pharmacists provide indispensable drug information to patients, however, few studies have examined the kind of information that patients want, or their satisfaction with pharmacists' advice. Therefore, we have examined a variety of factors, including individual patient characteristics, in order to identify ways to provide useful and appropriate drug information on an individual basis. In short, the aim of this study was to develop a drug information service that addresses patient needs. A prospective survey of patient satisfaction with the level of care provided by pharmacists, before and after a telephone counseling session, was performed over a 9 month period (2000-2001). Along with satisfaction ratings, the content of the interviews was assessed, along with patient characteristics. Correspondence analysis was used to classify the content of the consultations, and cluster analysis was performed to classify caller characteristics. In total, 2022 people were counseled. It should be noted that, on occasion, family members were interviewed instead of the patients themselves. The average counseling session was 11.5 minutes (n = 1876). Patients expressing the least dissatisfaction prior to counseling tended to have the highest satisfaction levels after counseling. Almost all patients were satisfied with the counseling they received. An association was found between levels of pre-counseling dissatisfaction and time spent counseling patients over the telephone. No dimensions of high inertia were identified by correspondence analysis of consultation content and patient characteristics (n = 1667), however, questions regarding "efficacy and indications", "dosages and administration", "anxiety of adverse events", "realization of adverse events", and "interactions" were deemed similar by correspondence analysis. This study shows that patient perceptions regarding drug information services differ from those of pharmacists. Furthermore, several subtypes of patients were identified, based on their responsiveness to counseling. Pharmacists should take this into consideration when standing face-to-face with patients in the setting of their pharmacy practice.

Tactile motion aftereffects produced by appropriate presentation for mechanoreceptors.

Tactile motion perception is one of the most important functions for realizing a delicate appreciation of the tactile world. To explore the neural dynamics of motion processing in the brain, the motion adaptation phenomenon can be a useful probe. Tactile motion aftereffects (MAE), however, have not been reported in a reproducible fashion, and the indistinctive outcomes of the previous studies can be ascribed to the non-optimal choice of adapting and testing stimuli. Considering the features of the stimuli used in the studies, the stimuli activated the different mechanoreceptors in the adapting and testing phase. Consequently, we tested tactile MAE using appropriate combinations of adapting and testing stimuli. We used three pins to generate sensation of apparent motion on the finger cushion. They were sequentially vibrated with the frequency of 30 Hz both in adapting and testing phases. It is expected that this procedure ensured stimulation for the same mechanoreceptor (Rapid-Adapting mechanoreceptor) in both the adaptation and test phases. Using this procedure, we found robust tactile MAEs in the various tactile motions such as the short-distance motion within the fingertip, the long-distance motion from the finger base to the fingertip, and the circular motion on the fingertip. Our development of a protocol that reliably produces tactile MAEs will provide a useful psychophysical probe into the neural mechanisms of tactile motion processing.